Novel oprf/i fusion proteins, their preparation and use

ABSTRACT

The present invention relates to a novel trimeric OprF/I fusion protein comprising a portion of the  Pseudomonas aeruginosa  outer membrane protein F which is fused with its carboxy terminal end to a portion of the amino terminal end of the  Pseudomonas aeruginosa  out membrane protein I, wherein said portion of the  Pseudomonas aeruginosa  outer membrane protein F comprises the amino acids 190-342 of SEQ ID NO: 1 and wherein said portion of the  Pseudomonas aeruginosa  outer membrane protein I comprises the amino acids 21-83 of SEQ ID NO: 2, and further to a novel Opr F/I fusion protein which contains a disulphide bond pattern, preferably selected from the group consisting of (a) Cys18-Cys27-bond, (b) Cys18-Cys27-bond and Cys33-Cys47-bond, and (c) Cys18-Cys47 and Cys27-Cys33-bond, and to immunogenic variants thereof having at least 85% identity to the amino acid sequence of SEQ ID NO: 3. The present invention also relates to a novel method for producing said OprF/I fusion proteins and to their use for the preparation of a pharmaceutical composition and for the preparation of antibodies or antibody derivatives which specifically bind said novel OprF/I fusion proteins.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/005,579, filed Oct. 16, 2013, now U.S. Pat. No. 9,359,412, which is anational stage filing under 35 U.S.C. §371 of international applicationPCT/EP2012/054783, filed Mar. 19, 2012, which was published under PCTArticle 21(2) in English, and claims the benefit under 35 U.S.C. §119(e)of U.S. provisional application Ser. No. 61/454,075, filed Mar. 18,2011, the disclosures of which are incorporated by reference herein intheir entireties.

FIELD OF THE INVENTION

The present invention relates to a novel trimeric OprF/I fusion proteincomprising a portion of the Pseudomonas aeruginosa outer membraneprotein F which is fused with its carboxy terminal end to a portion ofthe amino terminal end of the Pseudomonas aeruginosa outer membraneprotein I, wherein said portion of the Pseudomonas aeruginosa outermembrane protein F comprises the amino acids 190-342 of SEQ ID NO: 1 andwherein said portion of the Pseudomonas aeruginosa outer membraneprotein I comprises the amino acids 21-83 of SEQ ID NO: 2, and furtherto a novel OprF/I fusion protein which contains a disulphide bondpattern, preferably selected from the group consisting of (a)Cys18-Cys27-bond, (b) Cys18-Cys27-bond and Cys33-Cys47-bond, and (c)Cys18-Cys47 and Cys27-Cys33-bond, and to immunogenic variants thereofhaving at least 85% identity to the amino acid sequence of SEQ ID NO: 3.The present invention also relates to a novel method for producing saidOprF/I fusion proteins and to their use for the preparation of apharmaceutical composition and for the preparation of antibodies orantibody derivatives which specifically bind said novel OprF/I fusionproteins.

BACKGROUND OF THE INVENTION

Nosocomial infections are infections that are a result of treatment in ahospital or a healthcare service unit. Infections are considerednosocomial if they first appear 48 hours or more after hospitaladmission or within 30 days after discharge. This type of infection isalso known as a hospital-acquired infection (or, in generic terms,healthcare-associated infection). In the United States, the Center forDisease Control and Prevention estimates that roughly 1.7 millionhospital-associated infections, from all types of microorganism,including bacteria, combined, cause or contribute to 99,000 deaths eachyear. In Europe, where hospital surveys have been conducted, thecategory of Gram-negative infections are estimated to account fortwo-thirds of the 25,000 deaths each year. Nosocomial infections cancause severe pneumonia and infections of the urinary tract, bloodstreamand other parts of the body. Many types are difficult to attack withantibiotics, and antibiotic resistance is spreading to Gram-negativebacteria that can infect people outside the hospital.

In Gram-negative bacteria, lipopolysaccharides (LPS) and outer-membraneproteins are the major antigenic parts of the bacterial envelope. LPSbased vaccines have been extensively studied in the 1970s (Priebe G &Pier G., Vaccines for Pseudomonas aeruginosa 2003. New Bacterialvaccines, edited by Elfis R W, Brodeur B. 260-82). Parke Davis produceda vaccine Pseudogen from LPS of 7 different serogroups. Some activitywas observed with Pseudogen in non-randomized trials in cancer and burnpatients but not in cystic fibrosis (CF) and leukemia patients. BeingLPS based Pseudogen was very toxic and therefore not registered (Priebe,supra). Using two different versions of recombinant fusion proteins ofOpr's F and I, von Specht and colleagues have shown that activeimmunization can protect neutropenic mice and passive immunization canprotect SCID mice, both against a challenge dose 1000-fold above theLD50 (von Specht, B U et al., Protection of immunocompromised miceagainst lethal infection with Pseudomonas aeruginosa by active orpassive immunization with recombinant Pseudomonas aeruginosa outermembrane protein F and Outer membrane protein I fusion proteins. InfectImmun 1995; 63(5):1855-1862; Knapp B et al., A recombinant hybrid outermembrane protein for vaccination against Pseudomonas aeruginosa. Vaccine1999; 17(13-14):1663-1666). Said fusion protein was then tested forsafety and immunogenicity in healthy volunteers reaching high levels ofspecific serum antibodies. To achieve an enhanced mucosal immunogenicityin cystic fibrosis an emulgel formulation of said fusion protein wasdeveloped and tested for safety and immunogenicity in healthy volunteersand lung impaired patients. However, the serum antibody response wascomparatively low. A systemic i. m. booster has enhanced serum antibodyresponse as compared to solely mucosal vaccination schedule.

An outer membrane protein preparation composed of 4 different strains ofPseudomonas aeruginosa with a molecular weight range of 10-100 kDa wasdeveloped as a vaccine in Korea. The vaccine contained minimal amountsof polysaccharide and was tested in a double-blind, placebo-controlledtrial in burn patients (Jang II et al., Human immune response to aPseudomonas aeruginosa outer membrane protein vaccine. Vaccine 1999;17(2): 158-68). Antibody levels to the vaccine antigens rose by 2.3-foldin the placebo group (19 patients) and 4.9 fold in the vaccine group (76patients) (Kim D K et al., Comparison of two immunization schedules fora Pseudomonas aeruginosa outer membrane proteins vaccine in burnpatients. Vaccine 2001; 19(9-10):1274-83). Priebe and Pier criticizedthe study because the follow-up of patients in the trial was incomplete,analysis was not by intention-to-treat, and there were no data regardingclinical outcomes (Priebe, supra. A similar Opr vaccine was tested inRussia 10 years earlier (Stanislaysky E S et al., Clinico-immunologicaltrials of Pseudomonas aeruginosa vaccine. Vaccine 1991; 9(7):491-4).Pseudomonas aeruginosa vaccine (PV) containing predominantly cell-wallprotein protective antigens was tested for safety and immunogenicity byimmunization of 119 volunteers. The PV vaccine was well tolerated. Ahigh level of specific antibodies persisted for the 5-month period ofobservation. The antibody titers increased in 94-97% of volunteers andmoreover in 45.6% the antibody titers (the number of ELISA units)increased 2.5-3-fold and more. Anti-Pseudomonas aeruginosa plasma wasused for the treatment of 46 patients with severe forms of Pseudomonasaeruginosa infection (40 adults and six infants aged up to 2 years) and87% of the patients recovered. There have been no follow-up studies withthe PV vaccine after 1991.

Hospital-acquired infections are one of the major causes of death andserious illness worldwide, resulting in an annual cost burden of morethan USD 20 billion in the developed world. In the United States andEurope about 6 million patients become infected annually resulting in140,000 deaths per year. The incidence of nosocomial infections issteadily increasing due to increasing medical interventions andantibiotic resistance. Thus, minimizing risk of mortality throughhospital acquired infections by e.g. vaccination of burn victims andfibrosis patients, ICU patients and ventilated ICU patients is and isexpected to become even more so a major unmet medical need in saidpatients.

It has recently been found (US provisional application with applicationNo. 61/426,760) that a vaccine of the above-described hybrid fusionprotein comprising the Pseudomonas aeruginosa outer membrane protein I(Oprl or OMPI) which is fused with its amino terminal end to thecarboxy-terminal end of a carboxy-terminal portion of the Pseudomonasaeruginosa outer membrane protein F (OprF or OMPF) reduced the mortalityrate in mechanically ventilated intensive care patients significantlyover alum as placebo control. Mechanically ventilated intensive carepatients are at particular risk of acquiring severe and oftenlife-threatening forms of Pseudomonas aeruginosa or other infections,such as Ventilator-Associated Pneumonia (VAP), sepsis or soft tissueinfection. Such infections also may affect burn victims, severely burnedvictims, cancer and transplant patients who are immunosuppressed, andcystic fibrosis patients, Intensive Care Unit (ICU) patients orgenerally all hospitalized patients.

Generally, the expression of soluble OprF/I fusion protein in E. colileads to the formation of non immunological aggregates and misfoldedvariants. According to Worgall et al. (Worgall S et al., Protectionagainst P. aeruginosa with an adenovirus vector containing an OprFepitope in The Capsid., J. of Clinical Investigation, 2005, 115(5),1281-1289) it is assumed that the native OprF protein has one disulphidebridge from Cys200 to Cys209 of SEQ ID NO: 1 and two free cysteines atCys215 and Cys229 of SEQ ID NO: 1. In another publication (Rawling E Get al., Epitope Mapping of the Pseudomonas aeruginosa Major Outermembrane Protein OprF., Infection and Immunity, 1995, 63 (1), 38-42),however, two disulphide bonds from Cys200 to Cys209 and from Cys215 toCys229 of SEQ ID NO:1 are proposed. It cannot be expected that thereported disulphide bond pairing applies to the fusion protein OprF/Isince only amino acid No. 190 to amino acid No. 342 of SEQ ID NO: 1 fromthe native OprF protein are expressed. Since native OprF is an outermembrane protein and contains several transmembrane spans, it isexpected that folding in an aqueous environment differs from the foldedstructure of the natively expressed protein located in a membrane.

In addition, a pharmaceutical composition should be homogenous andstable. Thus, both good manufacturing practice as well as regulatoryauthority guidelines require that a dosage form of a pharmaceutical orpharmaceutical combination should be in the form of a homogeneousdispersion with respect to the active substances. There is a concern inthe field regarding aggregates and a potential for immunogenicity(Leonard J. Schiff, Biotechnology Products Derived from Mammalian CellLines: Impact of Manufacturing Changes (2004) Regulatory Affairs Focus,October 2004, pages 29-31).

SUMMARY OF THE INVENTION

In accordance with the present invention, it has now surprisingly beenfound that by a simple reduction and following reoxidation underspecific conditions an OprF/I fusion protein variant could be recovered.This specific variant shows a disulfide bond between Cys18 and Cys27 andtwo free cysteines at positions 33 and 47 (SEQ ID NO: 4) and a trimericstructure which has not been shown before.

Thus, in accordance with the particular findings of the presentinvention, there is provided:

-   1. An OprF/I fusion protein comprising a portion of the Pseudomonas    aeruginosa outer membrane protein F which is fused with its carboxy    terminal end to a portion of the amino terminal end of the    Pseudomonas aeruginosa out membrane protein I, wherein said portion    of the Pseudomonas aeruginosa outer membrane protein F comprises the    amino acids 190-342 of SEQ ID NO: 1 and wherein said portion of the    Pseudomonas aeruginosa outer membrane protein I comprises the amino    acids 21-83 of SEQ ID NO: 2, and further wherein said fusion protein    contains a disulphide bond pattern, preferably selected from the    group consisting of (a) Cys18-Cys27-bond (SEQ ID NO: 9), (b)    Cys18-Cys27-bond and Cys33-Cys47-bond (SEQ ID NO: 10), and (c)    Cys18-Cys47-bond and Cys27-Cys33-bond (SEQ ID NO: 11), or an    immunogenic variant thereof having at least 85%, preferably 90%, in    particular 95% identity to the amino acid sequence of SEQ ID NO: 4    and the same disulphide bond pattern as specified.-   2. A trimeric OprF/I fusion protein comprising a portion of the    Pseudomonas aeruginosa outer membrane protein F which is fused with    its carboxy terminal end to a portion of the amino terminal end of    the Pseudomonas aeruginosa out membrane protein I, wherein said    portion of the Pseudomonas aeruginosa outer membrane protein F    comprises the amino acids 190-342 of SEQ ID NO: 1 and wherein said    portion of the Pseudomonas aeruginosa outer membrane protein I    comprises the amino acids 21-83 of SEQ ID NO: 2, or an immunogenic    variant thereof having at least 85%, preferably 90%, in particular    95% identity to the amino acid sequence of SEQ ID NO: 4.-   3. A method for producing the OprF/I fusion protein as herein    described, said method comprising the steps of    -   (a) reducing said OprF/I fusion protein with a reducing agent,        preferably dithiothreitol (DTT), dithioerythritol (DTE) or        β-mercaptoethanol, and    -   (b) oxidizing the reduced OprF/I fusion protein with a redox        agent, preferably the redox agent glutathione        disulfide/glutathione or the redox agent cystine/cysteine, in        the presence of a reducing agent, preferably dithiothreitol        (DTT), dithioerythritol (DTE) or β-mercaptoethanol.-   4. A pharmaceutical composition, in particular a vaccine, comprising    said OprF/I hybrid.-   5. An antibody or antibody derivative which specifically binds said    OprF/I fusion protein.-   6. A pharmaceutical composition comprising said antibody or antibody    derivative which specifically binds said OprF/I fusion protein.

The invention will now be further illustrated below with the aid of theFigures, Tables, Sequence Listings and Examples, without beingrestricted hereto.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “about” means a general error range of +/−5%.

The term “immunogenic variant” means a sequence variant of the OprF/Ifusion protein which shows in vivo immunogenicity, e.g. in the BALB/cmouse model, e.g. have an ED50 value of 10 μg of lower, more preferablyan ED50 value of 5 μg or lower such as e.g. 4 μg or lower, 3 μg or loweror 2 μg or lower (see example section).

The term “binding specificity” or “specifically bind(s)” as used hereinrefers to the ability of an individual antibody combining site to reactwith only one antigenic determinant. The combining site of the antibodyis located in the Fab portion of the molecule and is constructed fromthe hypervariable regions of the heavy and light chains. Bindingaffinity of an antibody is the strength of the reaction between a singleantigenic determinant and a single combining site on the antibody. It isthe sum of the attractive and repulsive forces operating between theantigenic determinant and the combining site of the antibody. Specificbinding between two entities means a binding with an equilibriumconstant (KA) of at least 1×10⁷ M⁻¹, 10⁸ M⁻¹, 10⁹ M⁻¹, 10¹⁰ M⁻¹, 10¹¹M⁻¹, 10¹² M⁻¹, 10¹³ M¹. The phrase “specifically (or selectively) binds”to an antibody (e.g., an OprF/I agent-binding antibody) refers to abinding reaction that is determinative of the presence of an antigen(e.g., an OprF/I agent such as a trimer of a mixture of SEQ ID NOs: 9 to11) in e.g. a heterogeneous population of proteins and other compounds.In addition to the equilibrium constant (KA) noted above, an OprF/Iagent-binding antibody of the invention typically also has adissociation rate constant (Kd) of about 1×10⁻² s⁻¹, 1×10³ s⁻¹, 1×10⁴s⁻¹, 1×10⁴ s⁻¹, or lower, and binds to the OprF/I agent such as a trimerof a mixture of SEQ ID NOs: 9 to 11 with an affinity that is at least2-fold, 5-fold, 10-fold, 20-fold, 50-fold, preferably 100-fold, morepreferably 500-fold, or up to 1000-fold or more greater than itsaffinity for binding to a non-specific antigen. The phrases “an antibodyrecognizing an antigen” and “an antibody specific for an antigen” areused interchangeably herein with the term “an antibody which bindsspecifically to an antigen”.

Specific Aspects of the Invention

According to the present invention the OprF/I fusion protein containsparts of two outer membrane proteins of Pseudomonas aeruginosa,OprF₁₉₀₋₃₄₂ and OprI₂₁₋₈₃, and preferably an N-terminal tag which is inparticular useful for the better expression in a suitable host, e.g. E.coli, and/or purification of said fusion protein. After expression,OprF/I exists as heterogeneous mixture of misfolded forms (high and lowmolecular weight aggregates) caused by disulfide scrambling as shown inFIG. 1. Surprisingly, during purification it has been found that afterreduction and reoxidation of the fusion protein, novel disulfide bondswere created as shown in FIG. 8 resulting in a separable product mixtureof three main products (see FIGS. 7A and 7B, in particular peaks 1, 2and 3). Unexpectedly, the reoxidized fusion protein or fusion proteinmixture is stable and does not form undesired aggregates. Moreover, itwas unexpected that one of the three main products corresponding to peak1 shows the same disulfide bond and two blocked cysteines (caused bycovalent reaction with redox-agent (e.g. cysteines) used forreoxidation) as the native, not truncated OprF protein. However, notonly this specific fusion protein shows sufficient immunogenicity invivo but also the other two fusion protein variants corresponding topeaks 2 and 3 (see Table 3), which was indeed unexpected.

Therefore, one aspect of the present invention is directed to saidOprF/I fusion protein containing different disulphide bond patterns.Preferably the disulfide bond pattern corresponds to a singleCys18-Cys27-bond according to SEQ ID NO: 9. Another preferred disulphidebond pattern corresponds either to a Cys18-Cys27-bond and aCys33-Cys47-bond according to SEQ ID NO: 10, or to a Cys18-Cys47-bondand a Cys27-Cys33-bond according to SEQ ID NO: 11.

The described OprF/I fusion protein variants can either separately beisolated or as a mixture with or without further protein components, inparticular other fusion protein variants, preferably obtained after thepurification process described in the present specification. In case ofa mixture of the three main variants (peaks 1-3; FIG. 8), the relativedistribution of the variants in the purified mixture analyzed by RP-HPLCare: about 15% to about 18%, preferably about 16%, for the peak 1variant; about 67% to about 62%, preferably about 66%, for the peak 2variant; and about 18% to about 20%, preferably about 18%, for the peak3 variant (FIG. 9). In case of a mixture with further protein componentsas e.g. shown in FIGS. 7A and 7B, the total relative content or purityof all three main products (peaks 1-3) is at least about 75%, preferablyat least about 80% to about 90%, in particular at least about 85%, e.g.75% to 90% or 85% to 90%. The relative distribution of the three mainproducts in such mixture is the same as described above for a mixture ofonly the three main products. The specified values can be obtained e.g.by integration of the peak areas obtained by RP-HPLC at 280 and 214 nm

The present invention also encompasses an immunogenic variant of thedescribed OprF/I fusion protein which has at least 85%, preferably 90%,in particular 95% identity to the amino acid sequence of SEQ ID NO: 3with the proviso that the specified cysteine residues forming thedisulphide bonds are maintained.

In view of the above explanations, a particularly preferred embodimentof the present invention is a mixture, in particular a complex, ofOprF/I fusion proteins, each of the OprF/I fusion proteins comprises aportion of the Pseudomonas aeruginosa outer membrane protein F which isfused with its carboxy terminal end to a portion of the amino terminalend of the Pseudomonas aeruginosa out membrane protein I, wherein saidportion of the Pseudomonas aeruginosa outer membrane protein F comprisesthe amino acids 190-342 of SEQ ID NO: 1 and wherein said portion of thePseudomonas aeruginosa outer membrane protein I comprises the aminoacids 21-83 of SEQ ID NO: 2, said mixture containing, in particular inthe form of a trimer,

(a) an OprF/I fusion protein having only a Cys18-Cys27-bond (SEQ ID NO:9),

(b) an OprF/I fusion protein having a Cys18-Cys27-bond and aCys33-Cys47-bond (SEQ ID NO: 10), and/or

(c) an OprF/I fusion protein having a Cys18-Cys47-bond and aCys27-Cys33-bond (SEQ ID NO: 11).

The amino acid numbering is according to the amino acid sequence of SEQID NO: 4. The purity of said mixture is at least about 75%, preferablyat least about 80% to about 90%, in particular at least about 85%, e.g.75% to 90% or 85% to 90% compared to the whole protein content of themixture as preferably measured by RP-HPLC.

As explained above, a particular advantage of the present invention isthat the OprF/I fusion protein does not form undesired aggregates, inparticular high molecular weight aggregates, but preferably trimers.Interestingly, the OprF/I fusion protein trimers have a rather elongatedshape instead of a globular shape, and a high hydrodynamic radius, inparticular with a calculated Stokes-radius of 5.6 nm. The trimer wasstable in solution e.g. under physiological conditions such as e.g. pHaround 7 and room temperature, i.e. no dissociation was monitored.

Therefore, another aspect of the present invention is a trimeric OprF/Ifusion protein comprising a portion of the Pseudomonas aeruginosa outermembrane protein F which is fused with its carboxy terminal end to aportion of the amino terminal end of the Pseudomonas aeruginosa outermembrane protein I, wherein said portion of the Pseudomonas aeruginosaouter membrane protein F comprises the amino acids 190-342 of SEQ ID NO:1 and wherein said portion of the Pseudomonas aeruginosa outer membraneprotein I comprises the amino acids 21-83 of SEQ ID NO: 2, or animmunogenic variant thereof having at least 85%, preferably 90%, inparticular 95% identity to the amino acid sequence of SEQ ID NO: 3.

Preferably the trimeric OprF/I fusion protein possesses the samedisulfide bonds as explained above. In addition, the trimeric OprF/Ifusion protein(s) can be present in a mixture as also explained above.

Another embodiment of the present invention concerns the above-specifiedOprF/I fusion proteins which additionally contain a N-terminal tag.Therefore, the present invention also concerns a OprF/I fusion proteinwith 1-24 amino acids fused to its amino terminal end. Preferably theN-terminal tag is selected from Met-, Met-Ala-(His)₆- (SEQ ID NO: 5),Ala-(His)₆- (SEQ ID NO: 6),Met-Lys-Lys-Thr-Ala-Ile-Ala-Ile-Ala-Val-Ala-Leu-Ala-Gly-Phe-Ala-Thr-Val-Ala-Gln-Ala-(SEQID NO: 7),Met-Lys-Leu-Lys-Asn-Thr-Leu-Gly-Val-Val-Ile-Gly-Ser-Leu-Val-Ala-Ala-Ser-Ala-Met-Asn-Ala-Phe-Ala-(SEQID NO: 8), or any other N-terminal sequence disclosed in Table 1 ofGabelsberger et al. (1997) (Gabelsberger, J et al., A Hybrid OuterMembrane Protein Antigen for Vaccination Against Pseudomonas aeruginosa,Behring Inst. Mitt., 1997, 98, 302-314) namely the E. coli OmpT signalpeptide or the E. chrysanthemii PelB signal peptide. It is also possiblethat a spacer, preferably a Ser-Thr-Gly-Ser-spacer (SEQ ID NO: 12),between the tag and the N-terminus of the OprF/I fusion protein islocated. A particularly preferred OprF/I fusion protein contains anAla-(His)₆-N-terminus (SEQ ID NO: 6) because the fusion protein caneasily be purified by immobilized metal affinity chelate chromatographyas explained below.

In view of the above explanations, another particularly preferredembodiment of the present invention is, therefore, a mixture, inparticular a complex, of OprF/I fusion proteins, each of the OprF/Ifusion proteins comprises a portion of the Pseudomonas aeruginosa outermembrane protein F which is fused with its carboxy terminal end to aportion of the amino terminal end of the Pseudomonas aeruginosa outmembrane protein I, wherein said portion of the Pseudomonas aeruginosaouter membrane protein F comprises the amino acids 190-342 of SEQ ID NO:1 and wherein said portion of the Pseudomonas aeruginosa outer membraneprotein I comprises the amino acids 21-83 of SEQ ID NO: 2, and each ofthe OprF/I fusion proteins contains an Ala-(His)₆-N-terminus, saidmixture containing

(a) an OprF/I fusion protein having only a Cys18-Cys27-bond (SEQ ID NO:9),

(b) an OprF/I fusion protein having a Cys18-Cys27-bond and aCys33-Cys47-bond (SEQ ID NO: 10), and/or

(c) an OprF/I fusion protein having a Cys18-Cys47-bond and aCys27-Cys33-bond (SEQ ID NO: 11).

The amino acid numbering is according to the amino acid sequence of SEQID NO: 4. The purity of said mixture is at least about 75%, preferablyat least about 80% to about 90%, in particular at least about 85%, e.g.75% to 90% or 85% to 90% compared to the whole protein content of themixture as preferably measured by RP-HPLC as described above.Furthermore, the mixture contains preferably dimers and in particulartrimers of said OprF/I fusion protein.

Another aspect of the present invention concerns a method for producingthe above-specified OprF/I fusion protein(s). The preferred methodaccording to the present inventions comprises the steps of

-   (a) reducing said OprF/I fusion protein(s) with a reducing agent,    preferably dithiothreitol (DTT), dithioerythritol (DTE) or    β-mercaptoethanol, and-   (b) oxidizing the reduced OprF/I fusion protein(s) with a redox    agent, preferably the redox agent glutathione disulfide/glutathione    or the redox agent cystine/cysteine, in the presence of a reducing    agent, preferably dithiothreitol (DTT), dithioerythritol (DTE) or    β-mercaptoethanol.

The purpose of the reduction step is to break up all intra- andintermolecular disulfide bonds of highly cross-linked disulfideaggregates formed during expression in e.g. E. coli. Consequently, thefully reduced protein elutes as a single peak from a RP-HPLC column (seee.g. FIG. 2). The concentration of the reducing agent is in particularfrom about 3 mM to about 10 mM, preferably from about 3 mM to about 6mM, e.g. about 5 mM. DTT is the most preferred reducing agent because itis non-toxic. The reaction time of the reduction step (a) is inparticular from about 15 minutes to about 2 hours, preferably from about30 minutes to about 1 hour, especially about 30 minutes, and/or the pHvalue is preferably from about 7.0 to about 8.5, in particular about8.0.

The reoxidation can be carried out with different redox systems. Theprogress of reoxidation, i.e. the formation of disulfide bonds can bemonitored by RP-HPLC. Surprisingly it was found that in the presence ofreducing and oxidizing agent, in particular at low concentrations,reshuffling of the disulfide bonds resulted in essentially correct bondformation, i.e. misfolded forms of high and low molecular weightaggregates as e.g. shown in FIG. 1 were minimized and a stable solutionof immunogenic fusion proteins containing preferably dimers and inparticular trimers could be obtained (see e.g. FIGS. 2 and 3). Thefusion protein(s) are stable in aqueous solution at neutral pH in thepresence of a salt like NaCl, e.g. 0.15 M NaCl, e.g. the fusion proteinof SEQ ID NO: 4 in form as a trimer is stable for up to 24 monthsformulated in PBS at 2 to 8° C. The most preferred redox agent iscystine/cysteine and the most preferred reducing agent in thereoxidation step is DTT. The preferred concentration of the redox agentis from about 0.2 mM to about 4 mM, preferably about 0.2 mM to about 1mM, in particular about 0.2 mM to about 0.5 mM, and the concentration ofthe reducing agent is from about 0.5 mM to about 1.5 mM, preferablyabout 1 mM. The most preferred reoxidation of the fusion protein(s) canbe carried out in the presence of 0.5 mM cystine and 1 mM DTT finalconcentrations. The reaction temperature is in particular from about 18°C. to about 25° C., preferably at about 20° C. The reaction time of theoxidation step (b) is in particular from about 1 hour to about 20 hours,preferably from about 1 hour to about 6 hours, especially from about 1.5hours to about 2 hours, and/or the pH value is preferably from about 7.5to about 8.5, in particular about 8.0. Generally, a proteinconcentration from about 0.2 mg/mL to about 10 mg/mL, preferably fromabout 0.2 mg/mL to about 1 mg/mL, in particular from about 0.2 mg/mL toabout 0.5 mg/mL, especially at about 0.35 mg/mL is applicable.

Another preferred embodiment of the present invention concerns thesubsequent purification of the reoxidized fusion protein(s) by an anionexchange chromatography, in particular Diethylaminoethyl- (DEAE-),Diethyl-(2-hydroxypropyl)aminoethyl- (QAE-) or Trimethylaminomethyl-(Q-) exchange chromatography, preferably DEAE- and/or Q-exchangechromatography in order to reduce e.g. the endotoxin content and thegenomic DNA content. These remaining impurities can bind to anionexchange media at neutral to slightly basic pH even at higherconductivity, whereas the fusion protein product(s) remain in the flowthrough. It is most preferred to purify the reoxidized OprF/I fusionprotein(s) sequentially by DEAE- and Q-exchange chromatography,preferably by DEAE Sepharose® and Q-Sepharose®-HP chromatography,because the additional chromatography can separate between the variousforms of the fusion protein(s), e.g. peak 1, 2, 3, 4, 5, and highmolecular weight aggregates, and degradation by-products, e.g. a 7 kDfragment, which still may be present after the reoxidation and the firstchromatography purification step. Finally, the purified OprF/I fusionprotein(s) can be diafiltrated against a buffer solution, in particulara formulation buffer, e.g. an isotonic phosphate buffer saline solution(pH 7.4).

Generally, the above-described OprF/I fusion protein is produced byfermentation, preferably by expression in a suitable host, e.g. E. coli.Usually, the fusion protein is expressed intracellularly in soluble forme.g. at 30° C. and isolated after cell lysis with e.g. lysis buffercontaining e.g. high concentrations of a salt, e.g. NaCl, in particular0.5 M NaCl, and low concentration of a diazole e.g. imidazole, and inparticular 0.06 M imidazole. A preferred lysis buffer contains 0.1 MTris (pH 7.4), 0.5 M NaCl and 0.06 M imidazole.

Thereafter it is preferred to purify the OprF/I fusion protein byaffinity chromatography prior to the above-described reduction step.Preferred affinity chromatographies are immunoaffinity or immobilizedmetal ion affinity chromatography, in particular immobilized metal ionaffinity chromatography which can be used for capturing the His-taggedOprF/I fusion protein. Chelating Sepharose® loaded with copper ions ismost preferred. Thereafter, desalting e.g. on Sephadex G50 or byultra/diafiltration using a 100 kDa cut-off membrane is furtherpreferred in order to reduce the content of low molecular weightimpurities, e.g. imidazole or copper. In addition, a buffer change isconducted with this purification step. A preferred elution buffer is 0.1M Tris (pH 8.0) with 0.15M NaCl because this buffer is also a preferredbuffer for the following reduction and reoxidation steps. An overview ofthe most preferred production and purification process is shown in FIG.6. In short, the process can be summarized as follows:

-   (a) fermenting a suitable host, e.g. E. coli, expressing the    described OprF/I fusion protein,-   (b) lysing the host,-   (c) capturing the produced OprF/I fusion protein by affinity    chromatography, preferably by IMAC,-   (d) desalting the eluted OprF/I fusion protein,-   (e) reducing of the OprF/I fusion protein with a reducing agent,-   (f) reoxidizing the reduced OprF/I fusion protein with a redox agent    in the presence of a reducing agent,-   (g) purifying the reoxidized OprF/I fusion protein on anion exchange    chromatography, preferably on DEAE Sepharose®,-   (h) purifying the eluted OprF/I fusion protein on a further anion    exchange chromatography, preferably on Q-Sepharose®,-   (i) diafiltration the eluted OprF/I fusion protein into a    formulation buffer.

The formulation buffer is preferably an isotonic salt solution buffercontaining, e.g. KCl, NaCl and phosphate buffer (pH 7.4), as inparticular specified under the section “Materials”.

Consequently, the fusion protein(s) directly obtained by theabove-described methods is also a specific embodiment of the presentinvention. Examples of such fusion protein(s) are also described aboveand in the following examples.

Another aspect of the present invention is also a pharmaceuticalcomposition, in particular a vaccine, comprising the described OprF/Ifusion protein(s) or obtained by the above-described method(s), andoptionally at least one additive or adjuvant, in particular aluminiumhydroxide, which may serve as an additional stabilizer. A typicalformulation of the pharmaceutical composition contains an isotonicphosphate buffer saline solution (pH 7.4).

This preferred composition (SEQ ID NO:4 prepared according to the methoddescribed herein and formulated in PBS) is stable up to 24 months atabout 2° C. to about 8° C.

Another aspect of the present inventions concerns an antibody orantibody derivative which specifically binds the above-specified OprF/Ifusion protein(s) such as e.g. the trimer comprising the hereinspecified OprF/I fusion protein(s). The antibody is either polyclonal ormonoclonal, preferably it is a monoclonal antibody. The term “antibodyderivative” is understood as also meaning antigen-binding parts of theinventive antibody, prepared by genetic engineering and optionallymodified antibodies, such as, for example, chimeric antibodies,humanized antibodies, multifunctional antibodies, bi- or oligospecificantibodies, single-stranded antibodies, F(ab) or F(ab)₂ fragments, whichare all well known for a person skilled in the art.

The invention includes isolated antibodies and binding fragments thereofthat selectively bind trimers of OprF/I fusion proteins as describedherein. As used herein with respect to the binding of trimers of OprF/Ifusion proteins by the antibodies and binding fragments, “selectivelybinds” means that an antibody (binding fragment thereof) preferentiallybinds to a trimer of OprF/I fusion proteins (e.g., with greater avidityand/or binding affinity) than to an OprF/I fusion protein monomer. Inpreferred embodiments, the antibodies of the invention and bindingfragments thereof bind to a trimer of OprF/I fusion proteins with anavidity and/or binding affinity that is 1.1-fold, 1.2-fold, 1.3-fold,1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold,100-fold, 200-fold, 300-fold, 500-fold, 1000-fold or more than thatexhibited by the antibody and binding fragments thereof for an OprF/Ifusion protein monomer. Preferably, the antibody selectively bindstrimers of OprF/I fusion proteins, and not OprF/I fusion proteinmonomers, i.e., substantially exclusively binds to trimers of OprF/Ifusion proteins, or specifically binds trimers of OprF/I fusion proteinswithout substantial binding to OprF/I fusion protein monomers.

In some embodiment, the isolated antibodies or antigen-binding fragmentsthereof bind to a trimer-specific epitope. Generally, antibodies orantigen-binding fragments thereof that bind to a trimer-specific epitopepreferentially bind a trimer of OprF/I fusion proteins rather than aOprF/I fusion protein monomer. To determine if a selected antibody bindspreferentially (i.e., selectively and/or specifically) to a trimer ofOprF/I fusion proteins, each antibody can be tested in comparativeassays (e.g., a surface plasmon resonance (SPR) assay such as BiaCore orimmunoprecipitation followed by Western blotting) using trimers ofOprF/I fusion proteins and OprF/I fusion protein monomers. A comparisonof the results will indicate whether the antibodies bind preferentiallyto the trimer or to the monomer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically depicts the reduction and controlled reoxidationprocesses according to the present invention.

FIG. 2 shows the superimposition of RP-HPLC profiles of the OprF/Ifusion protein after expression and capturing on IMAC, after reduction,and after reoxidation/purification.

FIG. 3 shows the superimposition of SEC profiles of the OprF/I fusionprotein after expression and capturing on IMAC, and afterreoxidation/purification.

FIG. 4 shows the RP-HPLC analysis of the reoxidized IMAC/G50 pool.Samples were analyzed after 300 minutes and 21 hours.

FIG. 5 shows the change in retention time during SEC analysis of OprF/Ifusion protein samples at pH 8.0 and pH 2.

FIG. 6 shows a flow scheme of an exemplary production and purificationprocess of the OprF/I fusion protein.

FIG. 7A shows preparative RP-HPLC elution profiles; and FIG. 7B showsanalytical RP-HPLC elution profiles of an elected QSHP fraction.

FIG. 8 shows the disulphide bond pattern of peaks P1, P2 and P3 of theOprF/I fusion protein.

FIG. 9 shows the RP-HPLC peak pattern of purified OprF/I drug substancemixture.

SEQUENCES SEQ ID NO: 1 (full length Opr F) 1MKLKNTLGVV IGSLVAASAM NAFAQGQNSV EIEAFGKRYF TDSVRNMKNA DLYGGSIGYF 61LTDDVELALS YGEYHDVRGT YETGNKKVHG NLTSLDAIYH FGTPGVGLRP YVSAGLAHQN 121ITNINSDSQG RQQMTMANIG AGLKYYFTEN FFAKASLDGQ YGLEKRDNGH QGEWMAGLGV 181GFNFGGSKAA PAPEPVADVC SDSDNDGVCD NVDKCPDTPA NVTVDANGCP AVAEVVRVQL 241DVKFDFDKSK VKENSYADIK NLADFMKQYP STSTTVEGHT DSVGTDAYNQ KLSERRANAV 301RDVLVNEYGV EGGRVNAVGY GESRPVADNA TAEGRAINRR VEAEVEAEAKSEQ ID NO: 2 (precursor Opr I) 1MNNVLKFSAL ALAAVLATGC SSHSKETEAR LTATEDAAAR AQARADEAYR KADEALGAAQ 61KAQQTADEAN ERALRMLEKA SRK SEQ ID NO: 3 (OprF/I with N-tag plus Met) 1MAHHHHHHAP APEPVADVCS DSDNDGVCDN VDKCPDTPAN VTVDANGCPA VAEVVRVQLD 61VKFDFDKSKV KENSYADIKN LADFMKQYPS TSTTVEGHTD SVGTDAYNQK LSERRANAVR 121DVLVNEYGVE GGRVNAVGYG ESRPVADNAT AEGRAINRRV ESSHSKETEA RLTATEDAAA 181RAQARADEAY RKADEALGAA QKAQQTADEA NERALRMLEK ASRKSEQ ID NO: 4 (OprF/I with N-tag without Met) 1AHHHHHHAPA PEPVADVCSD SDNDGVCDNV DKCPDTPANV TVDANGCPAV AEVVRVQLDV 61KFDFDKSKVK ENSYADIKNL ADFMKQYPST STTVEGHTDS VGTDAYNQKL SERRANAVRD 121VLVNEYGVEG GRVNAVGYGE SRPVADNATA EGRAINRRVE SSHSKETEAR LTATEDAAAR 181AQARADEAYR KADEALGAAQ KAQQTADEAN ERALRMLEKA SRKSEQ ID NO: 5 (N-tag plus Met) 1 MAHHHHHHSEQ ID NO: 6 (N-tag without Met) 1 AHHHHHHSEQ ID NO: 7 (OmpA signal peptide E. coli) 1 MKKTAIAIAV ALAGFATVAQ ASEQ ID NO: 8 (OprF signal peptide P. aeruginosa) 1MKLKNTLGVV IGSLVAASAM AAFA SEQ ID NO: 9 (OprF/I with Cys18-Cys27-bond)Disulfide bond between Cys18 (underlined) and Cys27 (underlined) 1AHHHHHHAPA PEPVADVCSD SDNDGVCDNV DKCPDTPANV TVDANGCPAV AEVVRVQLDV 61KFDFDKSKVK ENSYADIKNL ADFMKQYPST STTVEGHTDS VGTDAYNQKL SERRANAVRD 121VLVNEYGVEG GRVNAVGYGE SRPVADNATA EGRAINRRVE SSHSKETEAR LTATEDAAAR 181AQARADEAYR KADEALGAAQ KAQQTADEAN ERALRMLEKA SRKSEQ ID NO: 10 (OprF/I with Cys18-Cys27-bond and a Cys33-Cys47-bond)Disulfide bond between Cys18-Cys27 (both underlined) and Cys33-Cys47 (both italic)1 AHHHHHHAPA PEPVADVCSD SDNDGVCDNV DKCPDTPANV TVDANGCPAV AEVVRVQLDV 61KFDFDKSKVK ENSYADIKNL ADFMKQYPST STTVEGHTDS VGTDAYNQKL SERRANAVRD 121VLVNEYGVEG GRVNAVGYGE SRPVADNATA EGRAINRRVE SSHSKETEAR LTATEDAAAR 181AQARADEAYR KADEALGAAQ KAQQTADEAN ERALRMLEKA SRKSEQ ID NO: 11 (OprF/I with Cys18-Cys47-bond and a Cys27-Cys33-bond)Disulfide bond between Cys18-Cys47 (both underlined) and Cys27-Cys33 (both italic)1 AHHHHHHAPA PEPVADVCSD SDNDGVCDNV DKCPDTPANV TVDANGCPAV AEVVRVQLDV 61KFDFDKSKVK ENSYADIKNL ADFMKQYPST STTVEGHTDS VGTDAYNQKL SERRANAVRD 121VLVNEYGVEG GRVNAVGYGE SRPVADNATA EGRAINRRVE SSHSKETEAR LTATEDAAAR 181AQARADEAYR KADEALGAAQ KAQQTADEAN ERALRMLEKA SRK SEQ ID NO: 12 (spacer) 1STGS

EXPERIMENTAL PART OF THE INVENTION Abbreviations

Abbreviation Explanation AUC Analytical ultracentrifugation CV Columnvolume DTT Dithiothreitol DV Diafiltration volumes DS Drug substanceED50 Reverse of the dilution of the samples resulting in 50%seroconversion rate EGT Eurogentec gDNA Genomic DNA GMT Geometric meantiter GSH Reduced glutathione GSSG Oxidized glutathione HCP Host cellprotein HPLC High performance liquid chromatography ICLL Intercell IMACImmobilized metal affinity chelate chromatography MALDI-ToF Matrixassisted Lased Desorption Ionization Mass Spectrometry-Time of FlightMALS Multi Angle Light Scattering β-ME Beta-mercaptoethanol PAGEPolyacrylamide gel Electrophoresis QSHP Q-Sepharose HP RP Reversed phaseRT Room temperature (about 20° C.) SCD Sedimentation CoefficientDistributions SEC Size exclusion chromatography UF/DFUltrafiltration/Diafiltration

Materials

NaOH (Riedel-de Haen), NaCl (Riedel-de Haen),Tris(hydroxymethyl)aminomethane (Merck KGaA, Darmstadt), L-Cystine(Aldrich), DTT (Sigma), HCl (Merck KGaA), Q-Sepharose® HP (GEHealthcare), DEAE-Sepharose® FF (GE Healthcare). All other materialswere of analytical grade if not otherwise stated.

Formulation buffer: Dulbecco's 1×PBS pH 7.4 (H15-002), lx concentrate(g/L)

KCl 0.2 g/L KH₂PO₄ 0.2 g/L NaCl 8.0 g/L Na₂HPO₄ anhydrous 1.15 g/L 

General Methods

Analytical RP-HPLC

Analytical RP-HPLC analysis of samples was performed on a Jupiter C4column (4.6 mm×150 mm, 300 A, 5 μm, Phenomenex) connected to a DionexUltimate 3000 HPLC system. Solvent A was water containing 0.1% TFA,solvent B was acetonitrile containing 0.1% TFA. Separation of peaks wasperformed by linear gradient elution from 27% B to 37% B in 13 min at aflow rate of 1 mL/min. The column temperature was set to 40° C. Peakdetection was performed at 214 nm and 280 nm.

For downstream development work an estimation of the specific OprF/Icontent in IMAC/G50 was necessary to calculate step yields. OprF/Icontent was determined by RP-HPLC. The HPLC system was calibrated withpurified, native (unreduced) OprF/I working standard. The proteincontent of the working standard was determined by UV 280 nm measurementbased on a calculated theoretical extinction coefficient for a 1 mg/mLsolution of ε_(0.1%)=0.373. Prior to analysis of IMAC/G50 pools byRP-HPLC, an aliquot was fully reduced by addition of DTT orβ-mercaptoethanol (100 mM final concentration) to split up the variousaggregated and misfolded (most probably disulfide scrambled) OprF/Ivariants. The samples were incubated at room temperature for 30 minutesand analyzed by RP-HPLC. After reduction, OprF/I eluted as a single peakcompared to the untreated IMAC/G50 pool. The content of reduced OprF/Iafter IMAC/G50 was calculated by integration of the peak area.

All other samples (e.g. reoxidized OprF/I, fractions from QS-HP etc.)were directly injected without further treatment and the OprF/Iconcentration was calculated.

Reoxidized samples can be immediately analyzed by RP-HPLC or formationof disulfide bonds can be quenched by acidification to pH 2-3 (˜20 μL 6%HCl per 1 ml reoxidation solution) and stored at 2-8° C. for subsequentanalysis.

Semi-Preparative RP-HPLC

Semi-Preparative RP-HPLC was used for isolation of individual peaksdetected by analytical RP-HPLC. Purification was done on a Jupiter C4column (10 mm×250 mm, 300 A, 5 μm, Phenomenex) connected to an ÄktaPurifier chromatography system. The stationary phase at preparativescale was the same as the one used at analytical scale. Solvent A waswater containing 0.1% TFA, solvent B was 80% acetonitrile in watercontaining 0.1% TFA. Sample volume was 2 to 4 mL (total protein load <2mg). Separation of peaks was performed by linear gradient elution from35% B to 40% B over 8 column volumes at a flow rate of 2.5 mL/min. Thecolumn temperature was set to 40° C. Peak detection was done at 280 and214 nm Fractions of 0.8 mL were collected and the pH was adjusted topH˜7 by addition of 0.25 mL 0.1 M sodium phosphate buffer, pH 7.0.Higher quantities (˜0.5 to 2 mg) of P1 to 4 were prepared by severalpreparative purification runs. After pooling of the desired fractionscontaining the individual peaks, samples were concentrated approximately5 times using a 5 kDa ultracentrifugation device (Millipore).Concentrated pools were desalted by PD10 columns (GE Healthcare) and thebuffer was exchanged against final drug product formulation buffer (1/10PBS diluted with 0.9% NaCl, pH ˜7). Final samples containing theisolated OprF/I variants were analyzed for purity and content by RP-HPLCand SEC-HPLC. The relative purity determined by RP-HPLC was at least90%. Samples were stored at −20° C. until further analysis.

SDS-PAGE

SDS-PAGE was done on 4-12% NuPAGE gels (Invitrogen) using MES runningbuffer. Samples were mixed with LDS sampling buffer under reducing ornon-reducing conditions and incubated for 5 min at 70° C. if nototherwise stated. Staining was done with colloidal Commassie or silverstain (Heukeshoven).

Western Blot Analysis

Western blotting was done with antibodies anti OprF/I 944/5 D5 epitope(1:20000 diluted) and 966/363 E3 epitope (1:10000 diluted).

pH and Conductivity Measurement

For determination of pH and conductivity of samples and buffers a WTW720 system was used. Conductivity was measured using the lineartemperature compensation mode at 25° C.

Endotoxin Measurement

Endotoxin measurement was done with a chromogenic LAL-assay (Cambrex).Selected samples were also measured in an external certified laboratorywith a conventional gel clot assay (Limulus Amoebocyte Lysate test).

Host Cell Protein Measurement (HCP)

For quantification of HCPs, a generic E. coli HCP ELISA kit (CygnusTechnologies, Inc.) was used.

Peptide-Mass Fingerprint and Disulphide Mapping

Purified fractions obtained from preparative RPC were analyzed byLC-MS/MS. Samples were digested with AspN or trypsin without reductionor after reduction and alkylation.

MALDI-ToF Mass Spectrometry

MALDI-ToF analysis was performed on a Voyager STR 4069 system (AppliedBiosystems). Sinapinic acid dissolved in 0.1% TFA/30% AcN was used assample matrix. DS samples were diluted five-fold with sample matrix and2 μl were placed on the target. A delayed extraction mode and positivepolarity was used. The system was externally calibrated with BSA (Masscalibration kit, Applied Biosystems). For internal calibration Myoglobin(Sigma M-0630, average Mr 16951.5) was spiked into DS samples at aconcentration of approximately 100 μg/mL. The mass accuracy for internalcalibration can be estimated with approximately ±0.3% (e.g. 24100±72Da), for external calibration ±0.6% (e.g. 24100±145 Da).

Native PAGE

The NativePAGE™ Novex® Bis-Tris Gel system is a near neutral pH,pre-cast polyacrylamide mini gel system to perform native(non-denaturing) electrophoresis. Native PAGE of OprF/I fusion proteinsamples was done on NativePAGE 4-16% Bis-Tris gels (Invitrogen)according to the manufacturers instruction. Sample buffer was 50 mMBisTris, 50 mM NaCl, 16 mM HCl, 10% w/v Glycerol, 0.001% Ponceau S, pH7.2. Running buffer was 50 mM BisTris, 50 mM Tricine, pH 6.8. Cathodebuffer was running buffer including 0.02% Coomassie G-250.

N-Terminal Sequencing

N-terminal sequencing was carried out using an Applied Biosystems 494HTmachine and the method of N-terminal Edman sequencing, where theN-terminal amino acid of the protein was sequentially removed chemicallyand identified by HPLC. The protein was first immobilized inside thesequencing instrument by either blotting it onto a PVDF membrane oradsorbing it onto a biobrene treated glass fibre filter. Subsequentlythe bound protein reacted with the Edman reagent, (phenylisothiocyanate,PITC) at high pH. After this reaction, the resulting compound wascleaved off the protein using anhydrous acid. The coupling and cleavageprocess was repeated for as many times as required. Usually 15 to 20amino acids (“amino acids” herein also referred to as “aa”) could beanalyzed. The cleaved products were converted to their stablephenylthiohydantoins, PTH, with aqueous acid, and then analyzed usingthe on-board HPLC. Identification of the amino acids was achieved bycomparing elution times compared to a standard mixture. Data from theHPLC was collected on a computer for visual calling of the sequence.

Alkylation of Thiolgroups

Free thiol groups in proteins can be detected by alkylation usingiodoacetamide, which reacts selectively with free thiol groups ofcysteines to produce carboxamidomethyl cysteine. If free thiol groupsare present, these would be covalently blocked resulting in a massincrease of 57 Da per attached iodacetamide molecule.

47 mg iodoacetamide were dissolved in 1 mL 1 M Tris-HCl, pH 8.0 (0.2 Miodoacetamide solution). 200 μL each of purified peak 1, 2 and 3(protein concentration approximately 200 μg/mL) were mixed with 20 μL ofiodoacetamide stock solution (final iodoacetamide concentration ˜0.02M).The OprF/I fusion protein sample (protein concentration approximately 1mg/mL) was 3 fold diluted with PBS to a final concentration ofapproximately 330 μg/mL. 30 μL iodacetamide stock solution were added to300 μL diluted DS. In another experiment the sample was reduced with 5mM DTT (20 min) before dilution and alkylation. All samples wereincubated at room temperature in the dark for 30 min followed by LC-MSanalysis.

Static Light Scattering Analysis

The chromatographic system consisted of an HPLC system from Dionexincluding an Ultimate 3000 pump and degasser, an Ultimate 3000autosampler and an Ultimate 3000 column compartment. Column andchromatographic conditions were the same as described for SEC-HPLC. Allsolvents were filtered through a 0.1 μm Supor Membrane filter (PallVacuCap 60). An injection volume of 100 μL was used for all samples ifnot stated otherwise.

Chromatographic detectors included a Dionex Ultimate 3000 photodiodearray detector set to 214 nm and 280 nm, a Shodex RI-101 refractiveindex detector and a DAWN TREOS MALS (multi angle light scattering)detector (Wyatt Technology Corporation), which was used in on-line mode.Chromatographic data collection and analysis was performed using theChromeleon software package (vers. 6.80, Dionex). Experimentalcollection and data analysis of the MALS-signals were performed with theASTRA software package (version 5.3.2.13, Wyatt Technology). Using thissoftware it was possible to collect and subsequently analyze the lightscattering signals (3 MALS angles) along with the UV-, and RI-signals.

Analytical Ultracentrifugation (AUC)

All experiments were performed with a BeckmanCoulter XL-I AnalyticalUltracentrifuge at 50.000 rpm and 25° C. Samples were placed insapphire-capped two-sector titanium centerpieces of 12 mm optical pathlength. 390 μL of solution and solvent were placed in the sample andreference sectors, respectively. Sedimentation traces were detected byrecording local differences in refractive index (interference optics).The samples were analyzed with a ten-fold dilution or without furtherdilution. Diffusion-corrected Sedimentation Coefficient Distributions(SCD) were calculated using the finite element approach proposed by P.Schuck, NIH (Peter Schuck et al., Biopolymers, Vol 54, Issue 5, pages328-341, October 2000). The frictional ratio f/f0 was treated as afitting variable. The density and viscosity of the buffer (phosphatebuffered saline, PBS) as well as the partial specific volume (v) of theproteins were calculated from composition with Sednterp. These valueswere used when calculating the respective SCD.

Analysis of OprF/I Fusion Protein Samples Including Aluminium Hydroxideby RP-HPLC

Aliquots (0.25 ml) of formulated OprF/I fusion protein were centrifugedat 16000×g for 10 minutes at 20° C. to separate the aluminium hydroxidesediment from the supernatant. The clear supernatant was removed andused for analysis of unbound fusion protein by RP-HPLC. The remainingpellet was resuspended in 0.25 ml of 0.1% TFA in water (pH ˜2). Sampleswere incubated at RT for 2 h, followed by 10 minutes centrifugation at16.000 g at room temperature to spin down the Aluminium particles. Theclear supernatant was used for analysis by RP-HPLC (TFA desorption).

Specific Methods and Results

Expression and Recovery of OprF/I Fusion Protein

OprF/I is a fusion protein of the pseudomonas outer membrane porinproteins OprF and OprI. It is expressed as a 224 aa hybrid proteincontaining a His₆-tag at its N-terminus. The N-terminal Met is cleavedoff after expression in E. coli. The primary structure of the expressedprotein (including the N-terminal methionine) is shown in SEQ ID NO: 3.

The molecular weight of the native protein has been calculated as24118.2 Da (full reduced protein, no N-terminal methionine). The pI hasbeen calculated as 5.3.

The protein of the present examples is a fusion protein of outermembrane protein F and I containing a N-terminal histidine tag (Histag). The protein was expressed in E. coli XL1-Blue/pTrc-Kan-OprF/I_Hisstrain. The OprF/I-His protein was expressed intracellularly in solubleform at 30° C.

Cell Lysis

OprF/I may be degraded by bacterial proteases, in particular when lysisbuffer without high concentration of NaCl and imidazole was used.Therefore, cells were resuspended in cold lysis buffer (1:5 dilution ofcell paste in buffer) consisting of 0.1 M Tris, pH 7.4, 0.5 M NaCl, 0.06M imidazole. Addition of 0.5 M NaCl particularly inhibited proteolyticdegradation of the molecule in the lysate. Resuspension and subsequenthomogenization (2 cycles at 800 bar) was done at cold room temperatureand the lysate was placed on ice immediately. Higher temperatures maylead to product degradation or higher protease activity.

IMAC-Copper Capture Step

Chelating Sepharose FF (loaded with copper ions) was used for capturingthe His-tagged OprF/I. After loading the lysate, elution was performedwith different concentrations of imidazole: 0.07 M, 0.325 M and 0.5 Mimidazole. OprF/I containing fractions elute at 0.325 M imidazole as twoseparate peaks. Analytical data showed that RP-HPLC elution profilecontained several peaks. If the same samples were analyzed under reducedconditions (addition of DTT or β-ME) only one major peak was observed.The various peaks in the untreated sample were most probably disulfidescrambled variants and aggregates of the native molecule.

An exemplary purification run was done with 992 g cell paste that isequivalent to 8.59 L of fermentation broth. After the IMAC purificationand desalting on Sephadex® G50 (see below) the total amount of OprF/Iwas approximately 1600 mg which is equivalent to 186 mg OprF/I per literfermentation broth.

Desalting on Sephadex G50

This step reduced the content of low molecular weight impurities (e.g.imidazole, copper, etc.) and a buffer exchange was conducted. Theloading volume was approximately 20% of the column volume. As elutionbuffer 0.1M Tris-HCl, 0.15M NaCl, pH 8.0 was used. It was the samebuffer used for reduction and reoxidation. Alternatively, this step wasalso replaced by UF/DF with a 100K cut-off membrane.

Reduction

After the IMAC/G50 steps, OprF/I exists as heterogeneous mixture ofmisfolded forms (high and low molecular weight aggregates) caused bydisulfide scrambling as schematically depicted in FIG. 1. Reduction ofdisulfide bonds was done with 5 mM DTT to break up all intra- andintermolecular disulfide bonds. The fully reduced protein elutes as asingle peak according to RP-HPLC data. DTT can be substituted by β-ME.Since DTT is not stable over a longer period of time in aqueoussolution, an aliquot of a freshly prepared DTT solution (1 M in water,used within 1 hour) is added to the IMAC/G50 pool under gentle stirring(5 mL of 1 M DTT stock solution per liter IMAC/G50 pool). The pool isincubated at room temperature for 30 minutes without stirring. Samplescan be analyzed by RP-HPLC to monitor the progress of reduction.

Reoxidation

For optimization of the reoxidation conditions, different redox systems(GSSG/GSH, cystamine/cysteamine, cystine/cysteine) were tested out inpresence of low concentration of DTT (1 mM) to allow correct reshufflingof the disulfide bond. The progress of reoxidation (formation ofdisulfide bonds) can be monitored by RP-HPLC after various timeintervals since the folding variants have different retention times.Reoxidation with cystamine/cysteamine was unsuccessful under the testedconditions. In a first set of experiments, GSSG and GSH were tested outas reoxidation agents. The reduced IMAC/G50 pool in 5 mM DTT was diluted5-fold into 0.1 M Tris-HCl, 0.15 M NaCl pH 8.0 containing GSSG (0-4 mM)under gentle stirring. DTT reacts with GSSG and forms GSH, GSSG andreduced/oxidized DTT. The final reoxidation conditions tested outcovered a broad range of different ratios of GSH, GSSG and DTT. Aliquotsof the samples were also quenched with HCl after various time intervalsand analyzed by RP-HPLC. At increasing GSSG concentration peak 1increases and peak 2 decreases. Formation of peak 1 occurs very early inthe reoxidation process and remains constant over time. The totalrecovery for peaks 1+2 was estimated to be ˜60% starting from thecompletely reduced protein (100%), the recovery of all detected peakswas approximately 90% compared to the starting material.

In a second set of experiments, cystine and cysteine were tested out asreoxidation agents. The reduced IMAC/G50 pool (5 mM DTT) was diluted5-fold into 0.1 M Tris-HCl, 0.15 M NaCl pH 8.0 containing variousconcentration of cystine (0-3 mM) and cysteine (0-3 mM). The final DTTconcentration was 1 mM. Please note that the 0.2 M stock solution ofcystine was prepared in 0.5 M NaOH. Samples were analyzed after 300 minand over night incubation at room temperature. No difference in RP-HPLCpeak pattern for each individual experiment between the two time pointswas observed except for the sample containing 1 mM DTT and no cystine.The protein was still reduced after 5 h, after over night incubationpeak 2 appeared. Depending on the final cystine and cysteineconcentration, different ratios of peak 1 and peak 2 were detected.RP-HPLC profiles showed that peak 1 concentration was sufficiently lowin presence of 0.5 mM cystine.

After preliminary studies of the various redox systems, it was decidedto use cystine as the oxidizing agent. During scale-up of the productionprocess for GMP production the concentration was further lowered to0.375 mM cystine. Representative RP-HPLC and SEC elution profiles priorand after reduction/reoxidation of IMAC/G50 pool are shown in FIG. 2 andFIG. 3. After reoxidation in presence of 0.5 mM cystine, the elutionprofiles observed by RP-HPLC and SEC were much more homogeneous comparedto the “untreated” IMAC/G50 pool. The various peaks, present in the IMACpool before reduction, shift to one major peak under reducingconditions. After reoxidation, one major peak (named as peak 2 in FIG.4) is observed with a different retention time compared to the reducedprotein. Peak 2 should represent the correctly folded OprF/I. Peak 2 issurrounded by three smaller peaks (peak 1, peak 3 and peak 4 in FIG. 4)that should be folding variants. Peaks eluting at approximately 13.17and 13.81 min, named as peak 5 and peak 6 in FIG. 4, are other foldingvariants (disulfide cross-linked aggregates according to MS data).

Further characterization of peak 1 by LC-MS showed an increase inmolecular weight of 240 Da compared to peak 2. This mass shift was mostprobably caused by covalent attachment of two molecules cysteine. Freecysteine was formed by the reaction of DTT with cystine, which resultedin 2 molecules cysteine. It was further discovered that peak 1 increaseswhile peak 2 decreases at increasing concentration of oxidizing agent(GSSG or cystine).

Evaluation of the main peak after reoxidation by SEC shows that theprotein does not exist as a monomer. The SEC column was calibrated withreference proteins (BioRad's size exclusion standard) ranging from 1.35to 670 kDa. The retention time of the main peak (˜25 min) corresponds toa calculated theoretical mass of ˜180 kDa under the assumption of aglobular shape and no unspecific interactions with the stationary phase.It was observed that this defined multimeric state was formedpreferential under the process and formulation conditions applied andseemed to be stable in aqueous solution at neutral pH in presence ofNaCl. At pH 7 to 8 the OprF/I fusion protein elutes as a multimercorresponding to 180 kDa, whereas in the acidified sample (pH ˜2) thepeak shifts to higher retention time (˜28 min) corresponding toapproximately 55 kDa (see FIG. 5). This change in retention time couldbe caused by dissociation of the multimer at low pH.

Purification by DEAE Sepharose FF

Additional purification of the OprF/I containing process stream by anionexchange chromatography after reoxidation was tested out to reduce thecontent of remaining endotoxins and gDNA. These remaining impuritieswould bind to anion exchange media at neutral to slightly basic pH evenat higher conductivity, whereas the product should remain in the flowthrough. DEAE Sepharose was tested out and found to have good propertiesto remove endotoxins without any major product losses by binding ofOprF/I onto the resin.

Purification by Q-Sepharose HP (QSHP)

After reoxidation and DEAE flow through chromatography, the proteinsolution was further purified by Q-Sepharose HP. Purification by QSHPresulted in an endotoxin concentration of ˜2 EU/mg in the main pool,which was within an acceptable low level.

Ultrafiltration/Diafiltration

Finally, the QS-HP pool was diafiltrated against formulation buffer(1×PBS buffer pH 7.4, Dulbecco, without Ca, Mg). A 10 kDa or 30 kDaregenerated cellulose membrane (Amicon Ultra 15 centrifugal filterdevice, Millipore), was used. OprF/I was detected in the permeate of the30 kDa membrane. Therefore, a 10 kDa membrane was used for final UF/DFinto formulation buffer resulting in a step yield of >95%. The pool wasadjusted to a final protein concentration of 1 mg/ml based on UVmeasurement.

An overview of the whole production and purification process is shown inFIG. 6. An overall yield of about 34% to about 40% of purified OprF/Ifusion protein was achieved.

Characterization of the Purified OprF/I Fusion Protein

Preparative Isolation of OprF/I Fusion Protein Variants

Selected side fractions from QSHP chromatography steps were used forpreparative isolation. A typical preparative elution profile andnomination of peaks detected is shown in FIGS. 7A and 7B. All combinedfractions containing the individual peaks were analyzed by SDS-PAGE andWestern blot under reducing and non-reducing conditions. Under reducingconditions all bands had similar migration properties compared to anOprF/I standard. Under non-reducing conditions, the content ofmultimeric OprF/I variants detected at approximately 60 kDa (calibratedagainst the molecular weight marker) increased for Peak C, D, 5 and 6.All bands were also detected by western blot analysis using monoclonalanti OprF/I antibodies. These results indicate that all peaks detectedby RP-HPLC are product related. This finding was also confirmed bypeptide-mass fingerprint analysis of the individual fractions. In finalDS only P1, 2, 3, 4 and 5 can be detected by RPC. The other peaks, A, B,C, D and 6, could be separated by preparative chromatography onQ-Sepharose HP from the main fractions. During Q-Sepharose HPchromatography a small peak eluted before the main peak. This fractioncontained a higher concentration of an OprF/I degradation product(denoted as 7 kDa peak) as detected by analytical RP-HPLC and MALDI-ToF.This peak was also shown to be a product related fragment consisting ofa 15.5 kDa and 7.2 kDa OprF/I fragment.

Analytical Characterization of OprF/I Fusion Protein Variants

The purified OprF/I fusion protein consists of different forms of themolecule as shown by RP-HPLC (see FIG. 4). Five peaks could be detectedby RP-HPLC. Peak 2 (P2) was the most prominent peak with a relativecontent of 50 to 55%, surrounded by peak 1 (P1), peak 3 (P3) and Peak 4(P4). Peak 5 (P5) was well separated from the other peaks eluting at aslightly higher retention time. The relative peak content is summarizedin Table 1. After reduction of the sample with β-ME or DTT, the elutionprofile changes. One major peak eluted and the individual variantsexhibited the same chromatographic retention time. Based on theseresults P1 to P4 are regarded as folding variants caused by differencesin disulphide bonding.

TABLE 1 Peak Sample 1 Sample 2 Sample 3 Sample 4 1 19% 14% 13% 11% 2 50%55% 54% 60% 3 18% 17% 19% 14% 4 9% 9% 9% 9% Sum of Peaks 87% 86% 86% 85%1, 2 and 3 Note: Reoxidation of sample 1 was done in presence of 0.5 mMcystine; samples 2, 3 and 4 were reoxidized in presence of 0.375 mMcystine. The slightly higher cystine concentration resulted in minorincrease in peak 1 content for sample 1.

MALDI-ToF Analysis

For MALDI-ToF analysis the system was calibrated externally against BSA.For internal calibration Myoglobin was used. All four samples showedsimilar mass spectra. The main signal was from native OprF/I monomerfollowed by OprF/I dimer and trimer peaks. Table 2 summarizes molecularmass obtained after internal calibration. Deviation from the expectedmolecular mass was within the experimental error (±0.3%). Mass peaks at24 kDa, 48 kDa and 72 kDa were detected, showing the presence of themonomeric, dimeric and trimeric OprF/I fusion proteins.

TABLE 2 Deviation from theoretical mass (Da)* (rel. % deviation fromPeak Analyzed mass (Da) theoretical MW) Monomer Sample 1 24096 −20(−0.08) Sample 2 24053 −63 (−0.26) Sample 3 24097 −19 (−0.08) Sample 424045 −71 (−0.30) Dimer Sample 1 48408 +176 (+0.36) Sample 2 48104 −128(−0.27) Sample 3 48239 −7 (−0.01) Sample 4 48031 −201 (−0.42) TrimerSample 1 72379 +31 (+0.04) Sample 2 72105 −243 (−0.34) Sample 3 72135−213 (−0.30) Sample 4 72250 −98 (−0.14) *theoretical mass: monomer 24114Da under the assumption of two disulfide bonds, dimer 48228, trimer72342

Native PAGE

Native PAGE of OprF/I fusion protein samples under non-reducing andreducing conditions were carried out as explained above. Bandintensities after Commassie blue staining were evaluated bydensitometry. Under native conditions one OprF/I main band was detectedin the range of approximately 180 kDa with a relative intensity ofapproximately 94 to 97%. Under reducing conditions the apparentmolecular size was determined as 206 kDa. The apparent molecular weightis in good correlation with SEC-HPLC data, but different from SEC-MALSand AUC results where OprF/I mass was in the range of 80 kDa (trimer).The separation mechanism for native PAGE is the same as for native SEC,separation properties strongly depend on the shape of the proteincomplex when it passes through the gel. This result confirms that OprF/Ihas a rather elongated shape with a high hydrodynamic radius.

N-Terminal Sequencing

The first 13 or 15 amino acids of two different samples were analyzed.No differences between the theoretical and detected amino acid sequencewere found. The sequencing results confirmed that the N-terminal Met wascompletely cleaved off during expression.

Alkylation of Thiolgroups

The results of the alkylation of the thiogroups of a OprF/I fusionprotein sample showed a mass increase after alkylation of +226 Dacorresponding to 4 attached molecules of iodacetamide (theoretical massincrease +228 Da; mass increase of +57 Da per attached iodacetamidemolecule). This result was expected since the reduced protein contains 4free cysteine residues. All other samples did not show an increase inmass. Based on these results peak P1 of the RP-HPLC (FIG. 4) could beconsidered as a twofold cysteinylated variant containing one additionaldisulphide bond. Peaks P2 and P3 were considered as variants containingtwo disulphide bonds.

Static Light Scattering (SEC/MALS)

SEC with refractive index/UV detection at 280 nm was combined with lightscattering for protein characterization and molecular weight detection.As the molar mass was constant over the cross section of the main peakeluting between 23 to 26 min, a defined monodisperse molecule specieseluted. For the main peak a molecular mass in the range of approx. 80 to86 kDa was detected. The cumulative mass fraction was in the range of 94to 98% (species 1).

The high molecular weight fraction (species 2) eluting between 20 to 22min showed a molecular mass in the range of 140 to 190 kDa. Due to thelow Rayleigh signal intensity for high molecular weight fraction themolecular mass determined exhibited a higher degree of variation. Thecumulative mass fraction of species 2 was in the range of 0.5 to 1% at arange between 120 to 200 kDa.

These results exhibit that OprF/I exists as a trimer (species 1) andthat only a small portion of the protein forms aggregates of highermolecular mass (species 2).

The results obtained by SEC-MALS are also in good correlation with AUCresults (see below). Results obtained by SEC/UV detection and nativePAGE indicated higher molecular masses for the OprF/I fusion protein inthe range of 180 kDa. Results obtained by SEC and native PAGE are basedon the assumption of a globular protein shape, whereas the protein shapedoes not influence static light scattering or AUC data. Based on theresults from the different methods that were applied, it was concludedthat the OprF/I trimer does not exist in a globular shape but exhibits alarge hydrodynamic radius.

Analytical Ultracentrifugation (AUC)

Sedimentation velocity profiles were recorded and deconvoluted withSedFit software to yield the sedimentation coefficient values of thesample components. The resulting calculated sedimentation coefficientand molecular mass for the individual species 1 (OprF/I fusion proteinmain peak) and species 2 (aggregates) were determined. The sedimentationcoefficient values for the dominant component species 1 agree ratherwell for all samples studied. This indicates that no significantdifferences exist between the different samples examined. The molar massof the main component species 1 differs within experimental variationfor this parameter. It generally indicates a trimer of the OprF/I fusionprotein. The molar masses of the monomer and trimer, as calculated fromthe sequence, are 24.1 kDa and 72.3 kDa, respectively.

No dissociation of this trimer occurred over the concentration rangeexamined. The Stokes-radius for the trimer was calculated to be 5.6 nm.The Stokes-radius for a globular protein of the expected trimer mass is2.8 nm. This indicates a highly asymmetrical and/or hydrated molecule.Species 2 appeared as a distinct peak at varying sedimentationcoefficients. This indicates that species 2 corresponds to a componentwith a distinct stoichiometry (hexamer, nonamer, etc.), as opposed tounspecific aggregation. These data are in very good correlation to theSEC-MALS results showing that the native OprF/I fusion protein exists asa trimer, but are significantly different from the calculated molecularmass obtained by SEC and native PAGE (overestimation of mass due tonon-globular shape). The primary and most reliable parameter from asedimentation velocity experiment is the sedimentation coefficientitself. For the calculation of the SCD, a single frictional coefficientwas assumed to apply for all sedimentation coefficients calculated. Itwas optimized in a fitting step. The frictional coefficient is necessaryfor the transformation of the SCD to a molar mass distribution (MMD). Inthe present study the signal for sedimentation coefficients <2 S onlyappeared at a ten-fold dilution. The possibility can be ruled that thispeak corresponds to a putative monomer of OprF/I out because species 1did not change. In conclusion, OprF/I is present in solution as atrimeric molecule. No dissociation occurred over the range ofconcentrations examined.

Disulfide Mapping

Disulphide Bond Mapping Using Nano-MS/MS Analysis

The aim of this study was to identify the differences in the disulphidebridge pattern between peaks 1, 2 and 3. The individual peaks wereisolated and enriched. The primary sequence contains 4 cystein residuesat position 18 (C1), 27 (C2), 33 (C3) and 47 (C4) (see SEQ ID NO: 3). Itwas concluded from the data of the intact molecular weight determinationby on-line LC/ES-MS that peak 1 has one disulphide bridge and twocysteinylations, and peaks 2 and 3 have two disulphide bridges. Thetryptic digest of all three peaks produced the peptide fragment 1 to 55,which contains all four cysteins of the protein. The observed masses forthis fragment in the three peaks confirmed the assignment from theintact MW analysis. The peptide fragment 1 to 55 from all three peakswere collected and subdigested with AspN and analysed by LC-MS. Based onthe interpretation of the raw data the structures according to FIG. 8were derived for the predominant component in the three different peaks.

These findings were confirmed by reduction and MS/MS experiments ofselected signals from the AspN subdigest. In addition to the disulphidebridge pattern deamidation was observed in the three different peaks. Inthe tryptic peptide 120 to 132, the Asn 124 is probably partlydeamidated. In different peptides, deamidation of Asn 45 was observed aswell.

Influence of Temperature on Stability

SDS-PAGE gels (reducing and non-reducing conditions) were run for OprF/Ifusion protein samples incubated at different temperatures over 10 days.Relative content of OprF/I fusion protein main band in reduced gels wascalculated by densitometric evaluation of the gels by normalization ofband intensities to 2-8° C. samples (reference). No degradation orchanges in band pattern were observed for samples stored at −80° C.,−20° C., 2-8° C. and RT (20° C.) over the storage period of 10 days.

Influence of pH on Stability

OprF/I fusion protein samples were incubated at different pH values atpH 1.98 to pH 11.1 and analyzed by RP-HPLC and SEC-HPLC. The main peakof the OprF/I fusion protein, which corresponds to the non-covalenttrimer, was constant with approximately 90% at pH 5.9 to 11.1 over thestorage period of at least 23 days at 2-8° C. The trimer reversiblydissociated at low pH (pH 2).

Aluminium Hydroxide as Additive/Adjuvant

RP-HPLC results showed that the OprF/I fusion protein could further bestabilized at pH 4.88 by binding onto aluminium hydroxide and could bedesorbed at high recoveries.

Immunogenicity of Different OprF/I Fusion Protein Fractions (BALB/cMouse Model)

Five BALB/c mice per group received 1 ml of different OprF/I fusionprotein fractions (peaks 1, 2 and 3 of obtained from semi-preparativeRP-HPLC fractions) and of the unfractionated OprF/I fusion protein (DS)i.p. at days 0 and 14. At day 21 the blood of the mice was tested forspecific antibodies and the values (GMT [μg/ml]+SD) determined atspecific doses (μg protein). The results are summarized in Table 3.

TABLE 3 dose Peak 1 Peak 2 Peak 3 DS 31.6 29.36 40.75 49.53 83.54 1015.58 4.59 24.63 31.04 3.16 0.09 0.03 0.24 0.70 1 0.01 0.01 0.01 0.050.316 0.01 0.01 0.01 0.01

It was concluded that all fractions as well as the unfractionated OprF/Ifusion protein induced specific antibodies. The ED50 value for the peak2 fraction has additionally been determined as 5.6 μg (unfractionatedOprF/I fusion protein: 1.8 μg).

CONCLUSIONS

-   1. The OprF/I fusion protein can be produced and purified without    cross-linked disulfide aggregates in an over all yield up to 40%    starting with the IMAC-Cu capture step (i.e. SEQ ID NO: 4 in the    form of a trimer wherein trimer content of more than about 90%    according to SEC and an aggregate content of less than 1%).-   2. The OprF/I fusion protein (SEQ ID NO: 4) produced in different    production lots is very consistent.-   3. The OprF/I fusion protein (SEQ ID NO: 4) exists as a trimer under    physiological conditions with a mean molecular mass of approximately    80 kDa and a relative content of 94 to 98%.-   4. The OprF/I fusion protein (SEQ ID NO: 4) produced according to    the present invention can be separated in several variants by    RP-HPLC (see FIGS. 4 and 8) Peak 1 (P1) is a two-fold cysteinylated    adduct at position 33 (C3) and 47 (C4) containing a disulphide bond    between position 18 (C1) and 27 (C2) (see also SEQ ID NO: 4). Peak 2    (P2) is a variant containing two disulphide bridges at positions 18    (C1)-27 (C2) and 33 (C3)-47 (C4). Peak 3 (P3) is a further variant    containing 2 disulphide bridges at positions 18 (C1)-48 (C4) and 27    (C2)-33 (C3).-   5. The OprF/I fusion protein (SEQ ID NO: 4) is stable from −80° C.    to +20° C. over a period of 10 days, and at pH 5.9 to 11.1 over a    period of 23 days at 2-8° C. Furthermore (data not shown), the    OprF/I fusion protein (SEQ ID NO: 4) is stable up to 24 months at 2    to 8° C. in PBS. At pH 4.88 the OprF/I fusion protein can be further    stabilized by binding onto aluminium hydroxide.-   6. All three variants (peaks 1, 2 and 3) as well as the    unfractionated OprF/I fusion protein induced specific antibodies    after vaccination of BALB/c mice.

Preferred Aspects

-   1. An OprF/I fusion protein comprising a portion of the Pseudomonas    aeruginosa outer membrane protein F which is fused with its carboxy    terminal end to a portion of the amino terminal end of the    Pseudomonas aeruginosa outer membrane protein I, wherein said    portion of the Pseudomonas aeruginosa outer membrane protein F    comprises the amino acids 190-342 of SEQ ID NO: 1 and wherein said    portion of the Pseudomonas aeruginosa outer membrane protein I    comprises the amino acids 21-83 of SEQ ID NO: 2, and further wherein    said fusion protein contains a disulphide bond pattern, preferably    selected from the group consisting of (a) Cys18-Cys27-bond (SEQ ID    NO: 9), (b) Cys18-Cys27-bond and Cys33-Cys47-bond (SEQ ID NO: 10),    and (c) Cys18-Cys47-bond and Cys27-Cys33-bond (SEQ ID NO: 11), or an    immunogenic variant thereof having at least 85%, preferably 90%, in    particular 95% identity to the amino acid sequence of SEQ ID NO: 4,    and the same disulphide bond pattern as specified.-   2. The OprF/I fusion protein according to aspect 1, wherein said    fusion protein is trimeric.-   3. The OprF/I fusion protein according to aspect 1 or 2, wherein    said fusion protein further contains 1-24 amino acids fused to its    amino terminal end, preferably selected from the group consisting of    Met-, Met-Ala-(His)₆- (SEQ ID NO: 5), Ala-(His)₆- (SEQ ID NO: 6),    Met-Lys-Lys-Thr-Ala-Ile-Ala-Ile-Ala-Val-Ala-Leu-Ala-Gly-Phe-Ala-Thr-Val-Ala-Gln-Ala-(SEQ    ID NO: 7),    Met-Lys-Leu-Lys-Asn-Thr-Leu-Gly-Val-Val-Ile-Gly-Ser-Leu-Val-Ala-Ala-Ser-Ala-Met-Asn-Ala-Phe-Ala-(SEQ    ID NO: 8), in particular Ala-(His)₆- (SEQ ID NO: 6).-   4. An OprF/I fusion protein mixture or complex containing or    consisting essentially of three OprF/I fusion proteins according to    aspect 1 or 3, in particular in the form of a trimer.-   5. The OprF/I fusion protein mixture or complex according to aspect    4, said mixture or complex containing or consisting essentially of    -   (a) an OprF/I fusion protein having only a Cys18-Cys27-bond (SEQ        ID NO: 9),    -   (b) an OprF/I fusion protein having a Cys18-Cys27-bond and a        Cys33-Cys47-bond (SEQ ID NO: 10), and/or    -   (c) an OprF/I fusion protein having a Cys18-Cys47-bond and a        Cys27-Cys33-bond (SEQ ID NO: 11).-   6. The OprF/I fusion protein mixture or complex according to aspect    5, wherein the relative distribution of the components are for    component (a) about 15% to about 18%, preferably about 16%; for    component (b) about 67% to about 62%, preferably about 66%; and for    component (c) about 18% to about 20%, preferably about 18%.-   7. The OprF/I fusion protein mixture or complex according to aspect    5 or 6, wherein the total relative content of all components (a)    to (c) compared to the total protein content is at least 75%,    preferably at least about 80% to about 90%, in particular at least    about 85%.-   8. The OprF/I fusion protein mixture or complex according to any of    aspects 5-7, wherein each of the OprF/I fusion proteins contains an    Ala-(His)₆-N-terminus, said mixture containing or consisting    essentially of, in particular in the form of a trimer,    -   (a) an OprF/I fusion protein having only a Cys18-Cys27-bond (SEQ        ID NO: 9),    -   (b) an OprF/I fusion protein having a Cys18-Cys27-bond and a        Cys33-Cys47-bond (SEQ ID NO: 10), and/or    -   (c) an OprF/I fusion protein having a Cys18-Cys47-bond and a        Cys27-Cys33-bond (SEQ ID NO: 11).-   9. A trimeric OprF/I fusion protein comprising a portion of the    Pseudomonas aeruginosa outer membrane protein F which is fused with    its carboxy terminal end to a portion of the amino terminal end of    the Pseudomonas aeruginosa out membrane protein I, wherein said    portion of the Pseudomonas aeruginosa outer membrane protein F    comprises the amino acids 190-342 of SEQ ID NO: 1 and wherein said    portion of the Pseudomonas aeruginosa outer membrane protein I    comprises the amino acids 21-83 of SEQ ID NO: 2, or an immunogenic    variant thereof having at least 85%, preferably 90%, in particular    95% identity to the amino acid sequence of SEQ ID NO: 3.-   10. The trimeric OprF/I fusion protein according to aspect 9,    wherein said fusion protein further contains 1-24 amino acids fused    to its amino terminal end, preferably selected from the group    consisting of Met-, Met-Ala-(His)₆- (SEQ ID NO: 5), Ala-(His)₆- (SEQ    ID NO: 6),    Met-Lys-Lys-Thr-Ala-Ile-Ala-Ile-Ala-Val-Ala-Leu-Ala-Gly-Phe-Ala-Thr-Val-Ala-Gln-Ala-(SEQ    ID NO: 7),    Met-Lys-Leu-Lys-Asn-Thr-Leu-Gly-Val-Val-Ile-Gly-Ser-Leu-Val-Ala-Ala-Ser-Ala-Met-Asn-Ala-Phe-Ala-(SEQ    ID NO: 8), in particular Ala-(His)₆- (SEQ ID NO: 6).-   11. A method for producing the OprF/I fusion protein according to    any of aspects 1-10, said method comprising the steps of    -   (a) reducing said OprF/I fusion protein with a reducing agent,        preferably dithiothreitol (DTT), dithioerythritol (DTE) or        β-mercaptoethanol, and    -   (b) oxidizing the reduced OprF/I fusion protein with a redox        agent, preferably the redox agent glutathione        disulfide/glutathione or the redox agent cystine/cysteine, in        the presence of a reducing agent, preferably dithiothreitol        (DTT), dithioerythritol (DTE) or β-mercaptoethanol.-   12. The method according to aspect 11, wherein in step (a) the    concentration of the reducing agent is from about 3 mM to about 10    mM, preferably from about 3 mM to about 6 mM.-   13. The method according to aspect 11 or 12, wherein in step (b) the    concentration of the redox agent is from about 0.2 mM to about 4 mM,    preferably about 0.2 mM to about 1 mM, in particular about 0.2 mM to    about 0.5 mM, and the concentration of the reducing agent is from    about 0.5 mM to about 1.5 mM, preferably about 1 mM.-   14. The method according to any of the aspects 11 to 13, wherein the    reaction temperature is from about 18° C. to about 25° C.,    preferably at about 20° C.-   15. The method according to any of the aspects 11 to 14, wherein the    reaction time of the reduction step (a) is from about 15 minutes to    about 2 hours, preferably from about 30 minutes to about 1 hour, in    particular about 30 minutes, and/or the pH value is from about 7.0    to about 8.5, in particular about 8.0.-   16. The method according to any of the aspects 11 to 15, wherein the    reaction time of the oxidation step (b) is from about 1 hour to    about 20 hours, preferably from about 1 hour to about 6 hours, in    particular from about 1.5 hours to about 2 hours, and/or the pH    value is from about 7.5 to about 8.5, in particular about 8.0.-   17. The method according to any of the aspects 11 to 16, wherein the    reoxidized OprF/I fusion protein is further purified by an anion    exchange chromatography, preferably Diethylaminoethyl- (DEAE-),    Diethyl-(2-hydroxypropyl)aminoethyl- (QAE-) or Trimethylaminomethyl-    (Q-) exchange chromatography, preferably DEAE- and/or Q-exchange    chromatography, in particular wherein the reoxidized OprF/I-fusion    protein is sequentially purified by DEAE- and Q-exchange    chromatography, preferably by DEAE Sepharose® and Q-Sepharose®    chromatography.-   18. The method according to any of the aspects 11 to 17, wherein    prior to the reduction of the OprF/I fusion protein, the OprF/I    fusion protein is purified by affinity chromatography, preferably by    immunoaffinity or immobilized metal ion affinity chromatography, in    particular by immobilized metal ion affinity chromatography.-   19. A pharmaceutical composition, in particular a vaccine,    comprising the OprF/I fusion protein according to any of the aspects    1 to 10 or obtained by the method according to any of the aspects 11    to 17, and optionally at least one additive or adjuvant, in    particular aluminium hydroxide, preferably formulated in an isotonic    phosphate buffer saline solution (pH 7.4).-   20. An antibody or antibody derivative which specifically binds the    OprF/I fusion protein according to any of the aspects 1 to 10 or    obtained by the method according to any of the aspects 11 to 17.-   21. A protein complex comprising three OprF/I fusion proteins of SEQ    ID NO: 4 or an immunogenic variant thereof having at least 85%,    preferably 90%, in particular 95% identity to the amino acid    sequence of SEQ ID NO: 4.-   22. A protein complex consisting at least 80%, preferably 85%, more    preferably 90% of three OprF/I fusion proteins of SEQ ID NO: 4 or an    immunogenic variant thereof having at least 85%, preferably 90%, in    particular 95% identity to the amino acid sequence of SEQ ID NO: 4.-   23. Complex of aspect 21 or 22, wherein the OprF/I fusion proteins    are selected from the group consisting of    -   (a) the OprF/I fusion protein of SEQ ID NO: 4 with a        Cys18-Cys27-bond (SEQ ID NO: 9), and    -   (b) the OprF/I fusion protein of SEQ ID NO: 4 with a        Cys18-Cys27-bond and Cys33-Cys47-bond (SEQ ID NO: 10), and    -   (c) the OprF/I fusion protein of SEQ ID NO: 4 with a        Cys18-Cys47-bond and Cys27-Cys33-bond (SEQ ID NO: 11),    -   or an immunogenic variant thereof having at least 85%,        preferably 90%, in particular 95% identity to the amino acid        sequence of SEQ ID NO: 4, and the same disulphide bond pattern        as specified in (a), (b) or (c).-   24. Complex of aspect 23, wherein the complex consists of a) about    15% to about 18% of the OprF/I fusion protein of SEQ ID NO: 4 with a    Cys18-Cys27-bond (SEQ ID NO: 9), b) about 62% to 67% of the OprF/I    fusion protein of SEQ ID NO: 4 with a Cys18-Cys27-bond and    Cys33-Cys47-bond (SEQ ID NO: 10), and c) about 18% to about 20% the    OprF/I fusion protein of SEQ ID NO: 4 with a Cys18-Cys47-bond and    Cys27-Cys33-bond (SEQ ID NO: 11).-   25. Complex of aspect 23, wherein the sum of a) the OprF/I fusion    protein of SEQ ID NO: 4 with a Cys18-Cys27-bond (SEQ ID NO: 9), b)    the OprF/I fusion protein of SEQ ID NO: 4 with a Cys18-Cys27-bond    and Cys33-Cys47-bond (SEQ ID NO: 10), and c) the OprF/I fusion    protein of SEQ ID NO: 4 with a Cys18-Cys47-bond and Cys27-Cys33-bond    (SEQ ID NO: 11) is equal or greater than 75%.-   26. Complex of aspect 21 or 22, wherein the OprF/I fusion proteins    are selected from the group consisting of    -   (a) the OprF/I fusion protein of SEQ ID NO: 4 with a        Cys18-Cys27-bond (SEQ ID NO: 9), or    -   (b) the OprF/I fusion protein of SEQ ID NO: 4 with a        Cys18-Cys27-bond and Cys33-Cys47-bond (SEQ ID NO: 10), or    -   (c) the OprF/I fusion protein of SEQ ID NO: 4 with a        Cys18-Cys47-bond and Cys27-Cys33-bond (SEQ ID NO: 11),    -   or an immunogenic variant thereof having at least 85%,        preferably 90%, in particular 95% identity to the amino acid        sequence of SEQ ID NO: 4, and the same disulphide bond pattern        as specified in (a), (b) or (c).-   27. Complex of aspect 21 or 22, wherein the OprF/I fusion proteins    is the OprF/I fusion protein of SEQ ID NO: 4 with a Cys18-Cys27-bond    (SEQ ID NO: 9) or an immunogenic variant thereof having at least    85%, preferably 90%, in particular 95% identity to the amino acid    sequence of SEQ ID NO: 4, and the same disulphide bond pattern as    specified in SEQ ID NO: 9.-   28. Complex of aspect 21 or 22, wherein the OprF/I fusion protein is    the OprF/I fusion protein of SEQ ID NO: 4 with a Cys18-Cys27-bond    and Cys33-Cys47-bond (SEQ ID NO: 10) or an immunogenic variant    thereof having at least 85%, preferably 90%, in particular 95%    identity to the amino acid sequence of SEQ ID NO: 4, and the same    disulphide bond pattern as specified in SEQ ID NO: 10.-   29. Complex of aspect 21 or 22, wherein the OprF/I fusion protein is    the OprF/I fusion protein of SEQ ID NO: 4 with a Cys18-Cys47-bond    and Cys27-Cys33-bond (SEQ ID NO: 11) or an immunogenic variant    thereof having at least 85%, preferably 90%, in particular 95%    identity to the amino acid sequence of SEQ ID NO: 4, and the same    disulphide bond pattern as specified in SEQ ID NO: 11.-   30. A pharmaceutical composition, in particular a vaccine,    comprising the protein complex according to any of the aspects 21 to    29 or the protein complex obtained by the method according to any of    the aspects 11 to 17, and optionally at least one additive or    adjuvant, in particular aluminium hydroxide, preferably formulated    in an isotonic phosphate buffer saline solution (pH 7.4).-   31. An antibody or antibody derivative which specifically binds the    protein complex according to any of the aspects 21 to 29 or the    protein complex obtained by the method according to any of the    aspects 11 to 17.-   32. The antibody or antibody derivative of aspect 31, wherein said    antibody or antibody derivative selectively binds to the protein    complex according to any of the aspects 21 to 29 or the protein    complex obtained by the method according to any of the aspects 11 to    17.-   33. The antibody or antibody derivative of aspect 31, wherein said    antibody or antibody derivative binds to a) the protein complex    according to any of the aspects 21 to 29 or b) the protein complex    obtained by the method according to any of the aspects 11 to 17 but    does not bind to a monomer of the OprF/I fusion protein according to    aspects 1 to 3.-   34. A pharmaceutical composition comprising the antibody or antibody    derivative according to aspects 32 or 33, and optionally    pharmaceutically acceptable excipients.-   35. The antibody or antibody derivative according to aspects 32 or    33 for use as a medicament, preferably for use in the reduction of    mortality.

1.-12. (canceled)
 13. An antibody or antibody derivative whichspecifically binds a protein complex comprising three OprF/I fusionproteins of SEQ ID NO: 4 or an immunogenic variant thereof having atleast 85% identity to the amino acid sequence of SEQ ID NO:
 4. 14. Anantibody or antibody derivative which selectively binds a proteincomplex comprising three OprF/I fusion proteins of SEQ ID NO: 4 or animmunogenic variant thereof having at least 85% identity to the aminoacid sequence of SEQ ID NO:
 4. 15. A pharmaceutical compositioncomprising the antibody or antibody derivative according to claim 13 andone or more pharmaceutically acceptable excipients.
 16. A pharmaceuticalcomposition comprising the antibody or antibody derivative according toclaim 14 and one or more pharmaceutically acceptable excipients.
 17. Theantibody or antibody derivative of claim 13, wherein the OprF/I fusionproteins are selected from the group consisting of (a) the OprF/I fusionprotein of SEQ ID NO: 4 with a Cys18-Cys27-bond (SEQ ID NO: 9), (b) theOprF/I fusion protein of SEQ ID NO: 4 with a Cys18-Cys27-bond andCys33-Cys47-bond (SEQ ID NO: 10), (c) the OprF/I fusion protein of SEQID NO: 4 with a Cys18-Cys47-bond and Cys27-Cys33-bond (SEQ ID NO: 11),and immunogenic variants thereof having at least 85%, preferably 90%, inparticular 95% identity to the amino acid sequence of SEQ ID NO: 4, andthe same disulphide bond pattern as specified in (a), (b) or (c). 18.The antibody or antibody derivative of claim 17, wherein the sum of a)the OprF/I fusion protein of SEQ ID NO: 4 with a Cys18-Cys27-bond (SEQID NO: 9), b) the OprF/I fusion protein of SEQ ID NO: 4 with aCys18-Cys27-bond and Cys33-Cys47-bond (SEQ ID NO: 10), and c) the OprF/Ifusion protein of SEQ ID NO: 4 with a Cys18-Cys47-bond andCys27-Cys33-bond (SEQ ID NO: 11) is equal or greater than 75%.
 19. Theantibody or antibody derivative of claim 14, wherein the OprF/I fusionproteins are selected from the group consisting of (a) the OprF/I fusionprotein of SEQ ID NO: 4 with a Cys18-Cys27-bond (SEQ ID NO: 9), (b) theOprF/I fusion protein of SEQ ID NO: 4 with a Cys18-Cys27-bond andCys33-Cys47-bond (SEQ ID NO: 10), (c) the OprF/I fusion protein of SEQID NO: 4 with a Cys18-Cys47-bond and Cys27-Cys33-bond (SEQ ID NO: 11),and immunogenic variants thereof having at least 85% identity to theamino acid sequence of SEQ ID NO: 4, and the same disulphide bondpattern as specified in either (a), (b) or (c).
 20. The antibody orantibody derivative of claim 19, wherein the sum of a) the OprF/I fusionprotein of SEQ ID NO: 4 with a Cys18-Cys27-bond (SEQ ID NO: 9), b) theOprF/I fusion protein of SEQ ID NO: 4 with a Cys18-Cys27-bond andCys33-Cys47-bond (SEQ ID NO: 10), and c) the OprF/I fusion protein ofSEQ ID NO: 4 with a Cys18-Cys47-bond and Cys27-Cys33-bond (SEQ ID NO:11) is equal or greater than 75%.
 21. The antibody or antibodyderivative of claim 13, wherein the OprF/I fusion proteins are theOprF/I fusion protein of SEQ ID NO: 4 with a Cys18-Cys27-bond (SEQ IDNO: 9) or an immunogenic variant thereof having at least 85%, preferably90%, in particular 95% identity to the amino acid sequence of SEQ ID NO:4, and the same disulphide bond pattern as specified in SEQ ID NO: 9.22. The antibody or antibody derivative of claim 13, wherein the OprF/Ifusion proteins are the OprF/I fusion protein of SEQ ID NO: 4 with aCys18-Cys27-bond and Cys33-Cys47-bond (SEQ ID NO: 10) or an immunogenicvariant thereof having at least 85%, preferably 90%, in particular 95%identity to the amino acid sequence of SEQ ID NO: 4, and the samedisulphide bond pattern as specified in SEQ ID NO:
 10. 23. The antibodyor antibody derivative of claim 13, wherein the OprF/I fusion proteinsare the OprF/I fusion protein of SEQ ID NO: 4 with a Cys18-Cys47-bondand Cys27-Cys33-bond (SEQ ID NO: 11) or an immunogenic variant thereofhaving at least 85%, preferably 90%, in particular 95% identity to theamino acid sequence of SEQ ID NO: 4, and the same disulphide bondpattern as specified in SEQ ID NO:
 11. 24. The antibody or antibodyderivative of claim 14, wherein the OprF/I fusion proteins are theOprF/I fusion protein of SEQ ID NO: 4 with a Cys18-Cys27-bond (SEQ IDNO: 9) or an immunogenic variant thereof having at least 85%, preferably90%, in particular 95% identity to the amino acid sequence of SEQ ID NO:4, and the same disulphide bond pattern as specified in SEQ ID NO: 9.25. The antibody or antibody derivative of claim 14, wherein the OprF/Ifusion proteins are the OprF/I fusion protein of SEQ ID NO: 4 with aCys18-Cys27-bond and Cys33-Cys47-bond (SEQ ID NO: 10) or an immunogenicvariant thereof having at least 85%, preferably 90%, in particular 95%identity to the amino acid sequence of SEQ ID NO: 4, and the samedisulphide bond pattern as specified in SEQ ID NO:
 10. 26. The antibodyor antibody derivative of claim 14, wherein the OprF/I fusion proteinsare the OprF/I fusion protein of SEQ ID NO: 4 with a Cys18-Cys47-bondand Cys27-Cys33-bond (SEQ ID NO: 11) or an immunogenic variant thereofhaving at least 85%, preferably 90%, in particular 95% identity to theamino acid sequence of SEQ ID NO: 4, and the same disulphide bondpattern as specified in SEQ ID NO: 11.